Activation of the prostaglandin FP receptor in human granulosa cells.
Prostaglandin F2 alpha (PGF2 alpha) has regulatory (mainly luteolytic) effects in the ovary but the mechanism of action is not completely understood. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques were used to demonstrate the presence of mRNA encoding the PGF2 alpha receptor (FP receptor) in human granulosa-lutein cells. Specific primers for the amplification of cDNA were designed and yielded a single product of 696 bp corresponding to the FP receptor. The identity of this product was verified by sequencing. Fluprostenol, a selective FP receptor agonist, activated phospholipase C (PLC) and increased intracellular free calcium concentration, confirming the functional activation of the receptor. We have demonstrated by Western blotting that granulosa cells express PLC-beta and PLC-gamma isoforms. The cells responded to pervanadate with increased PLC activity and increased tyrosine phosphorylation, demonstrating a functional PLC-gamma tyrosine kinase pathway. However, fluprostenol did not provoke any detectable tyrosine phosphorylation. Moreover, the effect of fluprostenol was inhibited through protein kinase C stimulation by phorbol 12, 13-dibutyrate, and was not affected when cells were treated with phenylarsine oxide, which blocks tyrosine phosphorylation. These results suggest that the FP receptor activates PLC-beta rather than PLC-gamma isoforms. Fluprostenol-induced activation was pertussis toxin resistant. Granulosa cells express G proteins of the Gq family (resistant to pertussis toxin) and mRNA for both G alpha q and G alpha 1 l has been identified by RT-PCR. In conclusion, human granulosa cells have a functional FP receptor the effects of which are mediated through PLC-beta activation probably via Gq/1 l.
Carrasco
M P
MP
Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, UK.
Asbóth
G
G
Phaneuf
S
S
López Bernal
A
A
D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D015237:Dinoprost / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D006107:Granulosa Cells / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008186:Luteolytic Agents / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011461:Prostaglandins F, Synthetic / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D011982:Receptors, Prostaglandin / Q000235:genetics / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
D007527:Isoenzymes; D008186:Luteolytic Agents; D011461:Prostaglandins F, Synthetic; D012333:RNA, Messenger; D011982:Receptors, Prostaglandin; C034671:prostaglandin F2alpha receptor; C006326:fluprostenol; D015237:Dinoprost; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium